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1.
PLoS One ; 15(9): e0233813, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903277

RESUMO

Since the initial report of glyphosate-resistant (GR) Amaranthus palmeri S. Watson in 2006, resistant populations have been reported in 28 states. The mechanism of resistance is amplification of a 399-kb extrachromosomal circular DNA, called the EPSPS replicon, and is unique to glyphosate-resistant plants. The replicon contains a single copy of the 10-kb 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene which causes the concomitant increased expression of EPSP synthase, the target enzyme of glyphosate. It is not known whether the resistance by this amplification mechanism evolved once and then spread across the country or evolved independently in several locations. To compare genomic representation and variation across the EPSPS replicon, whole genome shotgun sequencing (WGS) and mapping of sequences from both GR and susceptible (GS) biotypes to the replicon consensus sequence was performed. Sampling of GR biotypes from AZ, KS, GA, MD and DE and GS biotypes from AZ, KS and GA revealed complete contiguity and deep representation with sequences from GR plants, but lack of homogeneity and contiguity with breaks in coverage were observed with sequences from GS biotypes. The high sequence conservation among GR biotypes with very few polymorphisms which were widely distributed across the USA further supports the hypothesis that glyphosate resistance most likely originated from a single population. We show that the replicon from different populations was unique to GR plants and had similar levels of amplification.


Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Amaranthus/genética , Glicina/análogos & derivados , Herbicidas/metabolismo , Plantas Daninhas/genética , Amplificação de Genes , Glicina/metabolismo , Resistência a Herbicidas/genética , Proteínas de Plantas/genética , Replicon
2.
Sci Transl Med ; 12(555)2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32690628

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic, caused by infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is having a deleterious impact on health services and the global economy, highlighting the urgent need for an effective vaccine. Such a vaccine would need to rapidly confer protection after one or two doses and would need to be manufactured using components suitable for scale up. Here, we developed an Alphavirus-derived replicon RNA vaccine candidate, repRNA-CoV2S, encoding the SARS-CoV-2 spike (S) protein. The RNA replicons were formulated with lipid inorganic nanoparticles (LIONs) that were designed to enhance vaccine stability, delivery, and immunogenicity. We show that a single intramuscular injection of the LION/repRNA-CoV2S vaccine in mice elicited robust production of anti-SARS-CoV-2 S protein IgG antibody isotypes indicative of a type 1 T helper cell response. A prime/boost regimen induced potent T cell responses in mice including antigen-specific responses in the lung and spleen. Prime-only immunization of aged (17 months old) mice induced smaller immune responses compared to young mice, but this difference was abrogated by booster immunization. In nonhuman primates, prime-only immunization in one intramuscular injection site or prime/boost immunizations in five intramuscular injection sites elicited modest T cell responses and robust antibody responses. The antibody responses persisted for at least 70 days and neutralized SARS-CoV-2 at titers comparable to those in human serum samples collected from individuals convalescing from COVID-19. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection against SARS-CoV-2 infection.


Assuntos
Alphavirus/genética , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , RNA Viral/genética , Replicon/genética , Linfócitos T/imunologia , Vacinas Virais/imunologia , Animais , Formação de Anticorpos/imunologia , Infecções por Coronavirus/prevenção & controle , Compostos Inorgânicos/química , Lipídeos/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas/química , Pandemias , Primatas
3.
Viruses ; 12(6)2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32486283

RESUMO

Single-stranded positive RNA ((+) ssRNA) viruses include several important human pathogens. Some members are responsible for large outbreaks, such as Zika virus, West Nile virus, SARS-CoV, and SARS-CoV-2, while others are endemic, causing an enormous global health burden. Since vaccines or specific treatments are not available for most viral infections, the discovery of direct-acting antivirals (DAA) is an urgent need. Still, the low-throughput nature of and biosafety concerns related to traditional antiviral assays hinders the discovery of new inhibitors. With the advances of reverse genetics, reporter replicon systems have become an alternative tool for the screening of DAAs. Herein, we review decades of the use of (+) ssRNA viruses replicon systems for the discovery of antiviral agents. We summarize different strategies used to develop those systems, as well as highlight some of the most promising inhibitors identified by the method. Despite the genetic alterations introduced, reporter replicons have been shown to be reliable systems for screening and identification of viral replication inhibitors and, therefore, an important tool for the discovery of new DAAs.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas/métodos , Genes Reporter/fisiologia , Vírus de RNA/efeitos dos fármacos , Replicon/fisiologia , Animais , Antivirais/química , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Humanos , Vírus de RNA/genética , Transfecção , Células Vero
4.
Sci Rep ; 10(1): 7720, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32382084

RESUMO

Genome dynamics was investigated within natural populations of the soil bacterium Streptomyces. The exploration of a set of closely related strains isolated from micro-habitats of a forest soil exhibited a strong diversity of the terminal structures of the linear chromosome, i.e. terminal inverted repeats (TIRs). Large insertions, deletions and translocations could be observed along with evidence of transfer events between strains. In addition, the telomere and its cognate terminal protein complexes required for terminal replication and chromosome maintenance, were shown to be variable within the population probably reflecting telomere exchanges between the chromosome and other linear replicons (i.e., plasmids). Considering the close genetic relatedness of the strains, these data suggest that the terminal regions are prone to a high turnover due to a high recombination associated with extensive horizontal gene transfer.


Assuntos
Cromossomos Bacterianos/genética , Replicação do DNA/genética , Streptomyces/genética , Telômero/genética , Sequência de Aminoácidos/genética , Variação Genética , Plasmídeos/genética , Recombinação Genética/genética , Replicon/genética , Microbiologia do Solo , Sequências Repetidas Terminais/genética
5.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32404522

RESUMO

Autonomously replicating subgenomic Bungowannah virus (BuPV) RNAs (BuPV replicons) with deletions of the genome regions encoding the structural proteins C, ERNS, E1, and E2 were constructed on the basis of an infectious cDNA clone of BuPV. Nanoluciferase (Nluc) insertion was used to compare the replication efficiencies of all constructs after electroporation of in vitro-transcribed RNA from the different clones. Deletion of C, E1, E2, or the complete structural protein genome region (C-ERNS-E1-E2) prevented the production of infectious progeny virus, whereas deletion of ERNS still allowed the generation of infectious particles. However, those ΔERNS viral particles were defective in virus assembly and/or egress and could not be further propagated for more than three additional passages in porcine SK-6 cells. These "defective-in-third-cycle" BuPV ΔERNS mutants were subsequently used to express the classical swine fever virus envelope protein E2, the N-terminal domain of the Schmallenberg virus Gc protein, and the receptor binding domain of the Middle East respiratory syndrome coronavirus spike protein. The constructs could be efficiently complemented and further passaged in SK-6 cells constitutively expressing the BuPV ERNS protein. Importantly, BuPVs are able to infect a wide variety of target cell lines, allowing expression in a very wide host spectrum. Therefore, we suggest that packaged BuPV ΔERNS replicon particles have potential as broad-spectrum viral vectors.IMPORTANCE The proteins NPRO and ERNS are unique for the genus Pestivirus, but only NPRO has been demonstrated to be nonessential for in vitro growth. While this was also speculated for ERNS, it has always been previously shown that pestivirus replicons with deletions of the structural proteins ERNS, E1, or E2 did not produce any infectious progeny virus in susceptible host cells. Here, we demonstrated for the first time that BuPV ERNS is dispensable for the generation of infectious virus particles but still important for efficient passaging. The ERNS-defective BuPV particles showed clearly limited growth in cell culture but were capable of several rounds of infection, expression of foreign genes, and highly efficient trans-complementation to rescue virus replicon particles (VRPs). The noncytopathic characteristics and the absence of preexisting immunity to BuPV in human populations and livestock also provide a significant benefit for a possible use, e.g., as a vector vaccine platform.


Assuntos
Infecções por Pestivirus/virologia , Pestivirus/fisiologia , RNA Viral , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Deleção de Genes , Expressão Gênica , Genes Reporter , Engenharia Genética , Interações Hospedeiro-Patógeno , Infecções por Pestivirus/imunologia , Replicon , Proteínas do Envelope Viral/genética , Vírion , Montagem de Vírus
7.
Nat Commun ; 11(1): 2452, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415210

RESUMO

Many bacteria can exchange genetic material through horizontal gene transfer (HGT) mediated by plasmids and plasmid-borne transposable elements. Here, we study the population structure and dynamics of over 10,000 bacterial plasmids, by quantifying their genetic similarities and reconstructing a network based on their shared k-mer content. We use a community detection algorithm to assign plasmids into cliques, which correlate with plasmid gene content, bacterial host range, GC content, and existing classifications based on replicon and mobility (MOB) types. Further analysis of plasmid population structure allows us to uncover candidates for yet undescribed replicon genes, and to identify transposable elements as the main drivers of HGT at broad phylogenetic scales. Our work illustrates the potential of network-based analyses of the bacterial 'mobilome' and opens up the prospect of a natural, exhaustive classification framework for bacterial plasmids.


Assuntos
Bactérias/genética , Redes Reguladoras de Genes , Plasmídeos/genética , Algoritmos , Composição de Bases/genética , Bases de Dados Genéticas , Transferência Genética Horizontal/genética , Genes Bacterianos , Filogenia , Replicon/genética
8.
PLoS One ; 15(4): e0230857, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240216

RESUMO

The nucleotide sequences of plasmids pRC12 (12,342 bp; GC 43.99%) and pRC18 (18,664 bp; GC 34.33%), harbored by the bacteriocin-producer Lactobacillus curvatus CRL 705, were determined and analyzed. Plasmids pRC12 and pRC18 share a region with high DNA identity (> 83% identity between RepA, a Type II toxin-antitoxin system and a tyrosine integrase genes) and are stably maintained in their natural host L. curvatus CRL 705. Both plasmids are low copy number and belong to the theta-type replicating group. While pRC12 is a pUCL287-like plasmid that possesses iterons and the repA and repB genes for replication, pRC18 harbors a 168 amino acid replication protein affiliated to RepB, which was named RepB'. Plasmid pRC18 also possesses a pUCL287-like repA gene but it was disrupted by an 11 kb insertion element that contains RepB', several transposases/IS elements, and the lactocin Lac705 operon. An Escherichia coli / Lactobacillus shuttle vector, named plasmid p3B1, carrying the pRC18 replicon (i.e. repB' and replication origin), a chloramphenicol resistance gene and a pBluescript backbone, was constructed and used to define the host range of RepB'. Chloramphenicol-resistant transformants were obtained after electroporation of Lactobacillus plantarum CRL 691, Lactobacillus sakei 23K and a plasmid-cured derivative of L. curvatus CRL 705, but not of L. curvatus DSM 20019 or Lactococcus lactis NZ9000. Depending on the host, transformation efficiency ranged from 102 to 107 per µg of DNA; in the new hosts, the plasmid was relatively stable as 29-53% of recombinants kept it after cell growth for 100 generations in the absence of selective pressure. Plasmid p3B1 could therefore be used for cloning and functional studies in several Lactobacillus species.


Assuntos
Lactobacillus/genética , Plasmídeos/genética , Sequência de Aminoácidos/genética , Proteínas de Bactérias/genética , Sequência de Bases/genética , Replicação do DNA/genética , DNA Bacteriano/genética , Vetores Genéticos/genética , Origem de Replicação/genética , Replicon/genética , Análise de Sequência de DNA/métodos , Transposases/genética
9.
Science ; 368(6487)2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32217750

RESUMO

Transcription polymerases can exhibit an unusual mode of regenerating certain RNA templates from RNA, yielding systems that can replicate and evolve with RNA as the information carrier. Two classes of pathogenic RNAs (hepatitis delta virus in animals and viroids in plants) are copied by host transcription polymerases. Using in vitro RNA replication by the transcription polymerase of T7 bacteriophage as an experimental model, we identify hundreds of new replicating RNAs, define three mechanistic hallmarks of replication (subterminal de novo initiation, RNA shape-shifting, and interrupted rolling-circle synthesis), and describe emergence from DNA seeds as a mechanism for the origin of novel RNA replicons. These results inform models for the origins and replication of naturally occurring RNA genetic elements and suggest a means by which diverse RNA populations could be propagated as hereditary material in cellular contexts.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , RNA/biossíntese , Replicon , Transcrição Genética , Proteínas Virais/metabolismo , Biocatálise
10.
Nat Commun ; 11(1): 598, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001686

RESUMO

Metronidazole was until recently used as a first-line treatment for potentially life-threatening Clostridioides difficile (CD) infection. Although cases of metronidazole resistance have been documented, no clear mechanism for metronidazole resistance or a role for plasmids in antimicrobial resistance has been described for CD. Here, we report genome sequences of seven susceptible and sixteen resistant CD isolates from human and animal sources, including isolates from a patient with recurrent CD infection by a PCR ribotype (RT) 020 strain, which developed resistance to metronidazole over the course of treatment (minimal inhibitory concentration [MIC] = 8 mg L-1). Metronidazole resistance correlates with the presence of a 7-kb plasmid, pCD-METRO. pCD-METRO is present in toxigenic and non-toxigenic resistant (n = 23), but not susceptible (n = 563), isolates from multiple countries. Introduction of a pCD-METRO-derived vector into a susceptible strain increases the MIC 25-fold. Our finding of plasmid-mediated resistance can impact diagnostics and treatment of CD infections.


Assuntos
Clostridium difficile/fisiologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Metronidazol/farmacologia , Plasmídeos/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/microbiologia , Clostridium difficile/efeitos dos fármacos , Clostridium difficile/genética , Clostridium difficile/crescimento & desenvolvimento , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Fezes/microbiologia , Dosagem de Genes , Transferência Genética Horizontal/genética , Humanos , Metronidazol/uso terapêutico , Polimorfismo de Nucleotídeo Único/genética , Replicon/genética
11.
Nanomedicine ; 24: 102154, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31982617

RESUMO

In vivo delivery of large RNA molecules has significant implications for novel gene therapy, biologics delivery, and vaccine applications. We have developed cationic nanolipoprotein particles (NLPs) to enhance the complexation and delivery of large self-amplifying mRNAs (replicons) in vivo. NLPs are high-density lipoprotein (HDL) mimetics, comprised of a discoidal lipid bilayer stabilized by apolipoproteins that are readily functionalized to provide a versatile delivery platform. Herein, we systematically screened NLP assembly with a wide range of lipidic and apolipoprotein constituents, using biophysical metrics to identify lead candidates for in vivo RNA delivery. NLPs formulated with cationic lipids successfully complexed with RNA replicons encoding luciferase, provided measurable protection from RNase degradation, and promoted replicon in vivo expression. The NLP complexation of the replicon and in vivo transfection efficiency were further enhanced by modulating the type and percentage of cationic lipid, the ratio of cationic NLP to replicon, and by incorporating additive molecules.


Assuntos
Lipoproteínas HDL/metabolismo , RNA Mensageiro/metabolismo , Apolipoproteínas/química , Apolipoproteínas/metabolismo , Biomimética , Bicamadas Lipídicas/química , Lipoproteínas HDL/química , RNA Mensageiro/química , Replicon/genética
12.
Sci Rep ; 10(1): 36, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31913346

RESUMO

Extraintestinal multidrug resistant Escherichia coli sequence type (ST) 131 is a worldwide pandemic pathogen and a major cause of urinary tract and bloodstream infections. The role of this pandemic lineage in multidrug resistance plasmid dissemination is still scarce. We herein performed a meta-analysis on E. coli ST131 whole-genome sequence (WGS) databases to unravel ST131 plasmidome and specifically to decipher CTX-M encoding plasmids-clade associations. We mined 880 ST131 WGS data and proved that CTX-M-27-encoding IncF[F1:A2:B20] (Group1) plasmids are strictly found in clade C1, whereas CTX-M-15-encoding IncF[F2:A1:B-] (Group2) plasmids exist only in clade C2 suggesting strong plasmid-clade adaptations. Specific Col-like replicons (Col156, Col(MG828), and Col8282) were also found to be clade C1-associated. BLAST-based search revealed that Group1 and Group2 plasmids are narrow-host-range and restricted to E.coli. Among a collection of 20 newly sequenced Israeli ST131 CTX-M-encoding plasmids (2003-2016), Group1 and Group2 plasmids were dominant and associated with the expected clades. We found, for the first time in ST131, a CTX-M-15-encoding phage-like plasmid group (Group3) and followed its spread in the WGS data. This study offers a comprehensive way to decipher plasmid-bacterium associations and demonstrates that the CTX-M-encoding ST131 Group1 and Group2 plasmids are clade-restricted and presumably less transmissible, potentially contributing to ST131 clonal superiority.


Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli/genética , Genoma Bacteriano , Plasmídeos/genética , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/genética , Humanos , Pandemias , Filogenia , Replicon , Sequenciamento Completo do Genoma
13.
PLoS One ; 15(1): e0220484, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31990938

RESUMO

The growing occurrence of multidrug-resistant (MDR) Salmonella enterica in poultry has been reported with public health concern worldwide. We reported, recently, the occurrence of Escherichia coli and Salmonella enterica serovars carrying clinically relevant resistance genes in dairy cattle farms in the Wakiso District, Uganda, highlighting an urgent need to monitor food-producing animal environments. Here, we present the prevalence, antimicrobial resistance, and sequence type of 51 Salmonella isolates recovered from 379 environmental samples from chicken farms in Uganda. Among the Salmonella isolates, 32/51 (62.7%) were resistant to at least one antimicrobial, and 10/51 (19.6%) displayed multiple drug resistance. Through PCR, five replicon plasmids were identified among chicken Salmonella isolates including IncFIIS 17/51 (33.3%), IncI1α 12/51 (23.5%), IncP 8/51 (15.7%), IncX1 8/51 (15.7%), and IncX2 1/51 (2.0%). In addition, we identified two additional replicons through WGS (Whole Genome Sequencing; ColpVC and IncFIB). A significant seasonal difference between chicken sampling periods was observed (p = 0.0017). We conclude that MDR Salmonella highlights the risks posed to animals and humans. Implementing a robust, integrated surveillance system will aid in monitoring MDR zoonotic threats.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Genes Bacterianos , Plasmídeos/metabolismo , Doenças das Aves Domésticas/epidemiologia , Infecções por Salmonella/epidemiologia , Salmonella enterica/genética , Animais , Antibacterianos/classificação , Antibacterianos/farmacologia , Galinhas/microbiologia , Fazendas , Humanos , Vigilância Imunológica , Plasmídeos/química , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/transmissão , Prevalência , Replicon , Infecções por Salmonella/microbiologia , Infecções por Salmonella/transmissão , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/isolamento & purificação , Estações do Ano , Uganda/epidemiologia , Sequenciamento Completo do Genoma
14.
Proc Natl Acad Sci U S A ; 117(3): 1731-1741, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31896581

RESUMO

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and the leading cause for acute viral hepatitis worldwide. The virus is classified as a member of the genus Orthohepevirus A within the Hepeviridae family. Due to the absence of a robust cell culture model for HEV infection, the analysis of the viral life cycle, the development of effective antivirals and a vaccine is severely limited. In this study, we established a protocol based on the HEV genotype 3 p6 (Kernow C-1) and the human hepatoma cell lines HepG2 and HepG2/C3A with different media conditions to produce intracellular HEV cell culture-derived particles (HEVcc) with viral titers between 105 and 106 FFU/mL. Viral titers could be further enhanced by an HEV variant harboring a mutation in the RNA-dependent RNA polymerase. These HEVcc particles were characterized in density gradients and allowed the trans-complementation of subgenomic reporter HEV replicons. In addition, in vitro produced intracellular-derived particles were infectious in liver-humanized mice with high RNA copy numbers detectable in serum and feces. Efficient infection of primary human and swine hepatocytes using the developed protocol could be observed and was inhibited by ribavirin. Finally, RNA sequencing studies of HEV-infected primary human hepatocytes demonstrated a temporally structured transcriptional defense response. In conclusion, this robust cell culture model of HEV infection provides a powerful tool for studying viral-host interactions that should facilitate the discovery of antiviral drugs for this important zoonotic pathogen.


Assuntos
Vírus da Hepatite E/genética , Vírus da Hepatite E/fisiologia , Hepatite E/metabolismo , Hepatócitos/virologia , Animais , Antivirais/farmacologia , Carcinoma Hepatocelular , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Genótipo , Células Hep G2 , Hepatite E/virologia , Vírus da Hepatite E/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Camundongos , RNA Replicase/genética , RNA Replicase/metabolismo , Replicon , Ribavirina/metabolismo , Suínos , Carga Viral , Replicação Viral
15.
J Mol Biol ; 432(3): 745-761, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31931015

RESUMO

Bacterial genomes, organized intracellularly as nucleoids, are composed of the main chromosome coexisting with different types of secondary replicons. Secondary replicons are major drivers of bacterial adaptation by gene exchange. They are highly diverse in type and size, ranging from less than 2 to more than 1000 kb, and must integrate with bacterial physiology, including to the nucleoid dynamics, to limit detrimental costs leading to their counter-selection. We show that large DNA circles, whether from a natural plasmid or excised from the chromosome tend to localize in a dynamic manner in a zone separating the nucleoid from the cytoplasm at the edge of the nucleoid. This localization is in good agreement with silico simulations of DNA circles in the nucleoid volume. Subcellular positioning systems counteract this tendency, allowing replicons to enter the nucleoid space. In enterobacteria, these systems are found in replicons above 25 kb, defining the limit with small randomly segregated plasmids. Larger replicons carry at least one of the three described family of systems, ParAB, ParRM, and StbA. Replicons above 180 kb all carry a ParAB system, suggesting this system is specifically required in the cases of large replicons. Simulations demonstrated that replicon size profoundly affects localization, compaction, and dynamics of DNA circles in the nucleoid volume. The present work suggests that presence of partition systems on the larger plasmids or chromids is not only due to selection for accurate segregation but also to counteract their unmixing with the chromosome and consequent exclusion from the nucleoid.


Assuntos
Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , DNA Circular/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Replicon , Transporte Biológico , Plasmídeos/metabolismo
16.
Arch Virol ; 165(2): 331-343, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31832864

RESUMO

The most characteristic feature of the hepatitis C virus (HCV) genome in patients with chronic hepatitis C is its remarkable variability and diversity. To better understand this feature, we performed genetic analysis of HCV replicons recovered from two human hepatoma HuH-7-derived cell lines after 1, 3, 5, 7, and 9 years in culture: The cell lines 50-1 and sO harbored HCV 1B-1 and O strain-derived HCV replicons established in 2002 and 2003, respectively. The results revealed that genetic variations in both replicons accumulated in a time-dependent manner at a constant rate despite the maintenance of moderate diversity (less than 1.8% difference) between the clones and that the mutation rate in the 50-1 and sO replicons was 2.5 and 2.9 × 10-3 base substitutions/site/year, respectively. We found that the genetic distance of both replicons increased from 7.9% to 10.5% after 9 years in culture. In addition, we observed that the guanine + cytosine (GC) content of both replicon RNAs increased in a time-dependent manner, as observed in our previous studies. Finally, we demonstrated that the high sensitivity of both replicons to direct-acting antivirals was maintained even after 9 years in culture. Our results suggest that long-term cultured HCV replicon-harboring cells are a useful model for understanding the variability and diversity of the HCV genome and the drug sensitivity of HCV in patients with chronic hepatitis C.


Assuntos
Variação Genética/genética , Hepacivirus/genética , Replicação Viral/genética , Carcinoma Hepatocelular/virologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Genes Reporter/genética , Genoma Viral/genética , Genótipo , Hepatite C Crônica/virologia , Humanos , Neoplasias Hepáticas/virologia , RNA Viral/genética , Replicon/genética
17.
J Gen Virol ; 101(2): 188-197, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31859613

RESUMO

To establish infectious genotype 4a (GT4a) cell culture-derived hepatitis C virus (HCVcc), we constructed full-length ED43 and 12 mutants possessing single or double mutations that increase ED43 replicon replication, and performed cell culture after RNA transfection. Sequential long-term culture of full-length ED43 RNA-transfected cells showed increased viral production in two ED43 mutants named ED43 QK/SI and TR/SI among the tested clones. These ED43 mutants possessed a common mutation, R1405G, in the NS3 helicase region and another mutation, D2413G or V2414A, in the NS5a-NS5b cleavage site. Furthermore, serial reinfection of naïve Huh7.5.1 cells accelerated peak HCV production at an earlier time point after every infection. After the fourth infection, we found a common mutation, R1405G, and six additional mutations in both ED43 QK/SI and TR/SI mutants. All seven mutations supported continuous viral production for more than 40 days in both ED43 QS-7M (QK/SI with seven mutations) and ED43 TS-7M (TR/SI with seven mutations). In addition, ED43 TS-7M did not require additional mutations for continuous virus culture up to 124 days. Both ED43 QS-7M and TS-7M were sensitive to the neutralizing E2 antibodies HCV1 and AR3A and the direct-acting antivirals, simeprevir, ledipasvir and sofosbuvir. In conclusion, we established an infectious ED43 strain containing adaptive mutations, which is important for the analysis of HCV genotype-specific pathogenesis, development of pan-genotypic agents and analysis of drug resistance.


Assuntos
Hepacivirus/crescimento & desenvolvimento , Hepacivirus/genética , Mutação , Anticorpos Neutralizantes/farmacologia , Antivirais/farmacologia , Técnicas de Cultura de Células/métodos , Linhagem Celular , Genótipo , Hepacivirus/efeitos dos fármacos , Hepacivirus/imunologia , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Replicon/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Replicação Viral
18.
Proc Natl Acad Sci U S A ; 117(2): 1181-1190, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31879355

RESUMO

Negative-stranded/ambisense RNA viruses (NSVs) include not only dangerous pathogens of medical importance but also serious plant pathogens of agronomic importance. Tomato spotted wilt virus (TSWV) is one of the most important plant NSVs, infecting more than 1,000 plant species, and poses major threats to global food security. The segmented negative-stranded/ambisense RNA genomes of TSWV, however, have been a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from complementary DNA (cDNA) clones. First, a replication- and transcription-competent minigenome replication system was established based on 35S-driven constructs of the S(-)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by the 5' hammerhead and 3' ribozyme sequence of hepatitis delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA-dependent RNA polymerase (RdRp) gene. Next, a movement-competent minigenome replication system was developed based on M(-)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. Finally, infectious TSWV and derivatives carrying eGFP reporters were rescued in planta via simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(-)-gRNA, and L(+)-agRNA in which the glycoprotein gene sequence of M(-)-gRNA was optimized. Viral rescue occurred with the addition of various RNAi suppressors including P19, HcPro, and γb, but TSWV NSs interfered with the rescue of genomic RNA. This reverse genetics system for TSWV now allows detailed molecular genetic analysis of all aspects of viral infection cycle and pathogenicity.


Assuntos
DNA Complementar/genética , Tospovirus/genética , Tospovirus/fisiologia , Tospovirus/patogenicidade , RNA Polimerases Dirigidas por DNA/genética , Vírus Delta da Hepatite/genética , Proteínas do Nucleocapsídeo/genética , Doenças das Plantas/virologia , RNA Catalítico/genética , RNA Viral/genética , Replicon , Tabaco/virologia , Proteínas Virais/genética , Vírion/genética , Vírion/metabolismo , Replicação Viral
19.
Microbiol Immunol ; 64(4): 296-303, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31854467

RESUMO

The replicon system, which mimics viral genome replication in culture cells, has been widely used to analyze the genome replication of the hepatitis C virus (HCV). However, most HCV genomes used in the system include adaptive mutations (AMs) that are vital for replication in culture cells despite the nonexistence of such mutations in the genome of wild-type (WT) HCV in patients. In order to study the genome replications of WT HCV, new HCV subgenomic replicon (SGR) systems were established using Huh-7.5-derived cells producing Sec14-like protein 2 constitutively and SGR of KT9 (one of the HCV genotype 1b clones) with WT genome (SGR KT9WT) in this study. The replication efficiency and sensitivities of SGR KT9WT to anti-HCV drugs in the cloned cells permanently bearing replicon RNA, HS55-4 cells, were similar to those of reports using SGR, including AM. The SGR transient transfection system using SGR KT9WT and SGR KT9AM encoding secreted Nano-luciferase and HS55-4C cells established by the elimination of SGR KT9 RNA from HS55-4 cells, however, showed that the replication efficiency of SGR KT9WT was much lower than that of SGR KT9AM under a same condition. Furthermore, the sensitivities of SGR KT9WT to almost all tested anti-HCV reagents, except the inhibitor of miR-122, a cellular factor important for HCV replication, were quite low compared with SGR KT9AM. These results suggested that the new replicon systems might not only provide information about precise responses against new anti-HCV drugs but also reveal novel molecular mechanisms supporting negligent proliferation of HCV.


Assuntos
Farmacorresistência Viral , Hepacivirus/fisiologia , Replicação Viral , Antivirais/farmacologia , Linhagem Celular , Genoma Viral , Hepacivirus/genética , Humanos , Mutação , Replicon
20.
Proc Natl Acad Sci U S A ; 116(49): 24630-24638, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31744871

RESUMO

Herpesviruses must amplify their DNA to load viral particles and they do so in replication compartments. The development and functions of replication compartments during DNA amplification are poorly understood, though. Here we examine 2 functionally distinct replicons in the same cells to dissect DNA amplification within replication compartments. Using a combination of single-cell assays, computational modeling, and population approaches, we show that compartments initially were seeded by single genomes of Epstein-Barr virus (EBV). Their amplification subsequently took 13 to 14 h in individual cells during which their compartments occupied up to 30% of the nucleus and the nuclear volume grew by 50%. The compartmental volumes increased in proportion to the amount of DNA and viral replication proteins they contained. Each compartment synthesized similar levels of DNA, indicating that the total number of compartments determined the total levels of DNA amplification. Further, the amplification, which depended on the number of origins, was regulated differently early and late during the lytic phase; early during the lytic phase, the templates limited DNA synthesis, while later the templates were in excess, coinciding with a decline in levels of the viral replication protein, BMRF1, in the replication compartments. These findings show that replication compartments are factories in which EBV DNA amplification is both clonal and coordinated.


Assuntos
Replicação do DNA/genética , DNA Viral/biossíntese , Herpesvirus Humano 4/fisiologia , Replicon/genética , Replicação Viral/genética , Antígenos Virais/genética , Antígenos Virais/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Microscopia Intravital
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